This proposal entitled "Function of the insulin receptor substrate pp 185" focuses on the cloning expression and functional analysis of the putative insulin receptor (IR) substrate called ppl85. This work is scientifically and clinically important because diabetes is a contemporary health problem that affects about 2% of the world population. Whereas 10% of these individuals suffer from an absolute lack of insulin, most are diabetic because their cells do not respond fully to normal or elevated amounts of circulating insulin. The biochemical problems causing insulin resistance in these Type II diabetics are not well understood. In a few cases, mutations of the IR appear to be the culprit; however, rational treatments for the majority of diabetics requires a knowledge of the molecular details of insulin receptor signal transmission. The IR is a tyrosine-specific protein kinase which is essential for insulin signal transmission; however, physiologically important substrates for the IR kinase are uncharacterized. Previous studies initiated in this laboratory and elsewhere indicate that a protein of molecular weight 170-185 kDa (ppl85) is a ubiquitous substrate of the IR. However, the function of this protein and its role in insulin action is unknown. Recently in our laboratory, this rare protein was partially purified from insulin-- stimulated rat liver by affinity chromatography on immobilized antiphosphotyrosine antibodies. The primary amino acid sequences of several tryptic peptides from ppl85 were obtained, and the partial sequence data were used to prepare oligonucleotide probes which successfully identified 70% of the corresponding cDNA. The proposed experiments in this application build on this preliminary data and focus immediately in two productive directions: 1) the identification of the full-length cDNA clone of ppl85 from rat liver cDNA libraries and 2) the preparation of specific antibodies to verify that the isolated cDNA encodes the ppl85. Subsequently, additional studies are planned to identify the function of ppl85, and characterize its interactions with the insulin receptor. The specific tyrosine phosphorylation sites in ppl85 will be identified to define the substrate recognition site used by the IR. This information will be valuable for identification of additional receptor substrates. Finally, the cDNA of ppl85 will be overexpressed in cultured cells to determine its role in the mechanism of insulin action. Regions of ppl85 which are deduced from its primary amino acid sequence to be critical will be altered by site-directed mutagenesis to establish the importance of ppl85 in insulin action. This project will increase our understanding of insulin receptor signal transmission, provide insight into the continuing search for physiological substrates of the insulin receptor, and advance our understanding of the tyrosine kinase messenger system.